The broad objective of our lab is to understand the key mechanical and regulatory events that specify accurate chromosome segregation in human cells as it is directly relevant to human health, and drug development for cancer. We have focused on identifying and characterizing the molecular components of the kinetochore. The five aims of this proposal are to examine how some of these proteins work together to specify kinetochore microtubule attachments and to regulate the mitotic checkpoint. We propose to accomplish this by using molecular, biochemical and microscopic approaches to analyze the in vitro and in vivo activities of these proteins. Our analysis will include the roles of the moto proteins CENP-E and dynein/dynactin in kinetochore microtubule attachments and motility. We will investigate how the mitotic checkpoint monitors the activities of these two motors as a way to assess chromosome alignment. We will test whether CENP-E and hBUBR1 kinase are part of a mechanosensor complex that links kinetochore motility with the checkpoint. We study how and what activates the hBUBR1 kinase in mitosis and after spindle damage. We want to investigate whether interactions between dynein/dynactin and hZW10 and hROD proteins at kinetochores represent a second mechanosensor that links the checkpoint with dynein/dynactin functions at kinetochores. In addition to our efforts at the kinetochore, we want understand how the checkpoint blocks the Anaphase Promoting Complex from driving cells out of mitosis. We have isolated a factor from Hela cells that consists of hBUBR1, hBUB3, cdc20 and MAD2 that appears to be the physiologically relevant inhibitor of the Anaphase Promoting Complex. We propose to dissect the molecular interactions of the complex and determine the biochemical basis for how this complex binds and inhibits the APC in mitosis. Results from these studies should allow us to propose molecular models for how mechanical activities mediated by the kinetochore are linked with the checkpoint machinery.